Biomedical subjects

Erik J Fernandez

Publications and source records attributed to Erik J Fernandez.

16 recordsLinked to original sources

PubMed2007

Protein instability during HIC: hydrogen exchange labeling analysis and a framework for describing mobile and stationary phase effects.

Unfolding of marginally stable proteins is a significant factor in commercial application of hydrophobic interaction chromatography (HIC). In this work, hydrogen-deuterium isotope exchange labeling has been used to monitor protein unfolding on HIC media for different stationary phase hydrophobicities and as a function of ammonium sulfate concentration. Circular dichroism and Raman spectroscopy were also used to characterize the structural perturbations experienced by solution phase protein that had been exposed to media and by protein adsorbed on media. As expected, greater instability is seen on chromatographic media with greater apparent hydrophobicity. However, increased salt concentrations also led to more unfolding, despite the well-known stabilizing effect of ammonium sulfate in solution. A thermodynamic framework is proposed to account for the effects of salt on both adsorption and stability during hydrophobic chromatography. Using appropriate estimates of input quantities, analysis with the framework can explain how salt effects on stability in chromatographic systems may contrast with solution stability.

Chromatography, Agarose↗

PubMed2006

Loading, stationary phase, and salt effects during hydrophobic interaction chromatography: alpha-lactalbumin is stabilized at high loadings.

Amide hydrogen-deuterium exchange labeling has been used to study the effects of salt and protein loading on alpha-lactalbumin (BLA) stability during hydrophobic interaction chromatography (HIC). Stability in the adsorbed phase increased dramatically with increasing loading, and unfolding was nearly undetectable close to the resin saturation capacity. We also found that a butyl surface destabilized BLA more than a phenyl surface, despite the fact that BLA was bound more strongly on the phenyl surface. These observations have important implications for HIC process design and indicate that in some cases column capacity does not have to be sacrificed to preserve protein stability.

Adsorption↗

PubMed2006

Protein instability during HIC: describing the effects of mobile phase conditions on instability and chromatographic retention.

Hydrophobic interaction chromatography (HIC) is known to be potentially denaturing to proteins, but the effects of mobile phase conditions on chromatographic behavior are not well understood. In this study, we apply a model describing the effects of secondary protein unfolding equilibrium on chromatographic behavior, including the effects of salt concentration on both stability and adsorption. We use alpha-lactalbumin as a model protein that in the presence and absence of calcium, allows evaluation of adsorption parameters for folded and unfolded species independently. The HIC adsorption equilibrium under linear binding conditions and solution phase protein stability have been obtained from a combination of literature and new experiments. The effect of salt concentration on protein stability and the rate constant for unfolding on the chromatographic surface have been determined by fitting the model to isocratic chromatography data under marginally stable conditions. The model successfully describes the effects of added calcium and ammonium sulfate. The results demonstrate the importance of considering the effects on stability of mobile phase modifiers when applying HIC to marginally stable

Adsorption↗

PubMed2005

Mesoscopic simulation of adsorption of peptides in a hydrophobic chromatography system.

Mesoscopic simulations using Langevin dipoles on a lattice for the solvent and calculated partial charges for the solute have been used to estimate free energies of adsorption from data on reversed-phase chromatography on nine protected peptides covering a wide range of structures. There is a single parameter, the effective solvent dipole moment, that is fit to data for one peptide and used to predict properties of the other eight peptides. Good agreement of adsorption chemical potentials, including order of chromatographic retention times, is found for calculations that are Boltzmann-averaged over a set of orientations. In addition, the results suggest that there are preferential orientations for each peptide at the model hydrophobic chromatographic surface. Estimation methods for adsorption based on molecular descriptors and hydrophobicity scales are shown to be unreliable for these systems. With refinements and extensions, this simulation method should be applicable to solvents containing salt, such as in hydrophobic interaction chromatography, and to larger solutes including proteins.

Adsorption↗

PubMed2005

Analysis of column tortuosity for MnCl2 and bacterial diffusion using magnetic resonance imaging.

Subsurface bacteria often have to travel significant distances through tortuous porous media for purposes of groundwater remediation. In modeling such processes, motile bacteria are often represented as suspended colloids, ignoring their individual swimming or diffusive properties. In fact, bacterial migration is much more profoundly affected by the presence of porous media than is that of a chemical contaminant. In this study, we use magnetic resonance imaging (MRI) to perform noninvasive measurements of changes in bacterial concentration distributions across a packed column at a spatial resolution of 330 microm as a function of time. We analyze the diffusive behavior of Pseudomonas putida F1 under static conditions and compare that behavior to the diffusion of a chemical solute and of Escherichia coli NR50. Results indicate that P. putida cells experience a column tortuosity 50 times higher than that predicted from solute diffusion experiments. E. coli cells, which display shorter swimming run lengths in bulk solution than P. putida, seem to be less affected by the constricted pore space. Knudsen diffusion, or reductions in run length because of interactions between the diffusing bacteria and the porous media, may help to explain some of this discrepancy.

Diffusion↗

PubMed2004

Hydrophobic interaction chromatography selectivity changes among three stable proteins: conformation does not play a major role.

Interesting retention and selectivity changes have been noted for a number of proteins in hydrophobic interaction chromatography (HIC). In this study, we investigated the degree to which conformational changes may be responsible for selectivity changes of stable proteins. Hydrogen-deuterium isotope exchange detected by mass spectrometry was used to investigate changes in solvent accessibility during adsorption on HIC media. Lysozyme was determined to exhibit EX2 hydrogen exchange kinetics both in solution and adsorbed to Butyl Sepharose 4 Fast Flow and Phenyl Sepharose 6 Fast Flow high sub surfaces. A small, but significant, increase in solvent accessibility was observed upon adsorption. Similar approaches were used to analyze solvent accessibility of three stable proteins with melting temperatures above 50 degrees C exhibiting significant selectivity changes on Butyl Sepharose and Toyopearl Butyl 650M. While all three proteins (lysozyme, chymotrypsinogen A, and ovalbumin) exhibited enhanced exchange while adsorbed, no differences in solvent accessibility on the different adsorbents were observed. More detailed studies of lysozyme showed no significant changes in labeling prior or during elution. These results demonstrate that HIC surfaces examined here do not dramatically alter the structure of these stable proteins and that differences in conformation are not responsible for the selectivity changes observed. Thus, other factors such as different preferred binding orientations or variations between the media pore structure, size, and/or surface chemistry must be responsible.

Adsorption↗

PubMed2004

Quantification of bacterial chemotaxis in porous media using magnetic resonance imaging.

Bacterial chemotaxis has the potential to enhance biodegradation of organic contaminants in polluted groundwater systems. However, studies of bacterial chemotaxis in porous media are scarce. In this study we use magnetic resonance imaging (MRI) for the noninvasive measurement of changes in bacterial-density distributions in a packed column at a spatial resolution of 330 microm as a function of time. We analyze both the diffusive and the chemotactic behavior of Pseudomonas putida F1 in the presence of the chemical stimulus trichloroethylene (TCE). The migration of motile bacteria in experiments without TCE was described using an effective motility coefficient, whereas the presence of TCE required addition of a nonzero chemotactic sensitivity coefficient, indicating a significant response to TCE. The need for a chemotactic sensitivity term was justified by a test for statistical significance. This study represents the first quantification of bacterial chemotactic parameters within a packed column. For conditions under which chemotaxis occurs in porous media, it may potentially be exploited to significantly improve rates of in situ pollutant biodegradation in the subsurface environment, particularlyfor pollutants dissolved in water trapped in low-permeability formations or lenses.

Biodegradation, Environmental↗

PubMed2004

Benzyl alcohol-induced destabilization of interferon-gamma: a study by hydrogen-deuterium isotope exchange.

The destabilizing effect of a multidose preservative, benzyl alcohol, on IFN-gamma was investigated. Hydrogen-deuterium isotope exchange (HX) detected by mass spectrometry (MS) was used to detect tertiary structure changes and measure global unfolding rates. The experiments showed that tertiary structure changes previously reported using circular dichroism may involve only a limited portion of the protein with the hydrophobic core of the protein remaining intact. Protein unfolding rates measured by hydrogen exchange were very sensitive to benzyl alcohol concentration, and increased markedly when salt was also added. Dynamic light scattering and size-exclusion chromatography showed that a small fraction of the protein formed large aggregates during the first few days. Measurements at longer incubation times (up to 8 days) showed that a significant fraction of protein was trapped in a structure less protected from hydrogen exchange, but not completely unfolded. This fraction of protein may be responsible for the irreversible loss of activity observed in earlier studies.

Benzyl Alcohol↗

PubMed2003

Hydrogen exchange-mass spectrometry analysis of beta-amyloid peptide structure.

beta-Amyloid peptide (A beta) is the primary protein component of senile plaques in Alzheimer's disease and is believed to be responsible for the neurodegeneration associated with the disease. A beta has proven to be toxic only when aggregated; however, the structure of the aggregated species associated with toxicity is unknown. In the present study, we use hydrogen-deuterium isotope exchange (HX)-electrospray ionization mass spectrometry (MS) along with enzymatic digestion as a tool to examine at near residue level, the changes in A beta structure associated with aggregation to a fibril form. Our results show that the structure of A beta intermediate species formed early in the course of fibrillogenesis is dependent upon solvent conditions. Additionally, the HX-MS data of peptic A beta fragments suggest that the C-terminal segment of the peptide is approximately 35% protected from exchange in fibril-containing samples, relative to monomeric A beta species prepared in DMSO/H(2)O. The N-terminus (residues 1-4) is completely unprotected from exchange, and the fragment containing residues 5-19 is over 50% protected from exchange in the fibril-containing samples. This work contributes to our understanding of A beta structure associated with aggregation and toxicity and further application of this approach may aid in the design of agents that intervene in the A beta aggregation processes associated with neurotoxicity.

Amyloid beta-Peptides↗

PubMed2003

Aprotinin conformational distributions during reversed-phase liquid chromatography. Analysis by hydrogen-exchange mass spectrometry.

Hydrogen-exchange mass spectrometry analysis of the stable protein aprotinin during reversed-phase liquid chromatography shows both native and unfolded protein. The behavior is consistent with only two conformational states, a near-native state and a fully solvent-accessible state, with reversible interchange of species within and between the mobile and stationary phases. The amount of unfolded form is greater on C18 relative to C4 alkyl modified silica surfaces. The addition of (NH4)2SO4, Na2SO4, NaCl, or NaSCN to the mobile phase stabilized native conformation on the chromatographic surface, especially on the C4 media. Finally, the retention and the proportion of denatured form increases with added salts in anorder consistent with the lyotropic series, but reversed from that observed for small molecules.

Aprotinin↗

PubMed2003

Alpha-lactalbumin tertiary structure changes on hydrophobic interaction chromatography surfaces.

Hydrogen exchange (HX) detected by mass spectrometry (MS) was used to analyze the structure of calcium-free alpha-lactalbumin, a model protein with marginal stability. Two chromatographic peaks were observed from samples of pure protein eluted from SOURCE phenyl hydrophobic interaction chromatography (HIC) media. Whole-protein HX measurements showed that the less-retained peak had solvent exposure similar to native protein in the absence of the HIC surface while the retained protein was nearly, although not fully, solvent exposed. The formation of these two peaks was kinetically limited. The protein also refolded successfully following elution. In addition, proteolytic fragmentation was used to analyze HX at the peptide level. This approach revealed that helix C was the most stable region of alpha-lactalbumin under native conditions and in the flow-through peak. Helix C also formed the core of residual native structure in the partially unfolded protein in the retained peak. The results suggest that residues that are most solvent accessible under native conditions may be those most likely to unfold upon adsorption.

Amino Acid Sequence↗

PubMed2003

Analysis of bacterial random motility in a porous medium using magnetic resonance imaging and immunomagnetic labeling.

In this study, we demonstrate the application of immunomagnetic labeling and magnetic resonance imaging (MRI) for the noninvasive visualization of changes in bacterial density distributions as a function of time in a water-saturated porous medium. Magnetite particles (50-60 nm diameter) were attached via a monoclonal antibody to the surface' of Escherichia coli K12 NR50 cells. The cells maintained their motility after labeling, and the presence of the magnetite did not significantly alter cell swimming speed. Diffusive migration for both motile and nonmotile E. coli through a porous medium with a particle-diameter distribution of 250-300 microm was compared. The movement of the nonmotile cells was described by an effective random motility coefficient consistent with Brownian diffusion of a nonmotile colloid. An effective coefficient determined a priori from bacterial motility in an aqueous medium and properties of the porous medium adequately described the movement of the motile cells. The ability to noninvasively visualize bacterial concentrations within an opaque porous medium in real time provides researchers with a powerful tool for studying bacterial transport in porous media. This is important for understanding the impact of bacterial transport on remediation strategies for environmental cleanup of polluted groundwater.

Antibodies, Monoclonal↗

PubMed2002

Structural features of interferon-gamma aggregation revealed by hydrogen exchange.

Using hydrogen-deuterium exchange (HX) and electrospray ionization mass spectrometry, we have investigated the stability and structural changes of recombinant human interferon-gamma (IFN-gamma) during aggregation induced by guanidine hydrochloride (GdnHCl) and potassium thiocyanate. First, HX labeling was initiated after the amorphous aggregates were formed to probe the tertiary structure of the aggregated state. Second, labeling was performed at low protein concentrations to assess stability under aggregation prone conditions. In 1 M GdnHCl, the stability of IFN-gamma was greatly reduced and much less protection from HX in solution was observed. Exchange under these conditions was slower in helix C than in the rest of the protein. Aggregates formed in 1 M GdnHCl showed a HX pattern consistent with a partially unfolded state with an intact helix C. Although aggregates formed in 0.3 M KSCN exhibited a HX pattern similar to those formed in GdnHCl, the solution phase HX pattern in 0.3 M KSCN was surprisingly comparable to that of the native state. Varying the aggregation time before performing HX revealed that KSCN first precipitated native protein and then facilitated partial unfolding of the precipitated protein. These results show that helix C, which forms the hydrophobic core of the IFN-gamma dimer, is highly protected from HX under native conditions, is more stable in GdnHCl than the rest of the protein and remains intact in both GdnHCl- and KSCN-induced aggregates. This suggests that native-state HX patterns may presage regions of the protein susceptible to unfolding during aggregation.

Deuterium↗

PubMedDate not supplied

Toward a robust model of packing and scale-up for chromatographic beds. 1. Mechanical compression.

The packing of compressible biochromatographic resins at large scale suffers from a poor understanding of how column packing method, resin properties, and column geometry impact column performance. To improve understanding, we develop and evaluate a one-dimensional, continuum mechanics model of column packing by mechanical compression. We show that the model can quantitatively predict the change in bed height, applied stress, and internal axial porosity profile without adjustable parameters when the modulus and wall friction coefficients are determined independently. The model possesses theoretical relationships for wall support and resin rigidity that should enable it to describe the mechanical compression of any biochromatographic resin for any column diameter. Moreover, this framework could provide a path to analogous models for flow packing and dynamic axial compression.

Chromatography, Liquid↗

PubMedDate not supplied

Toward a robust model of packing and scale-up for chromatographic beds. 2. Flow packing.

We developed and evaluated a model for predicting the flow packing of nonrigid chromatographic resins. The model is based on elasticity theory and accounts for resin rigidity and column diameter. When a modulus determined from a standard mechanical compression (consolidation) test is used, the model captures the primary phenomena of the scale-up process. However, moduli determined from flow-packing experiments improve the accuracy of the predictions and show that the apparent rigidity of chromatographic resins is lower for flow packing than for mechanical compression. Using a modulus from flow-packing experiments provided quantitative scale-up predictions of flow packing carried out in columns with diameters between 200 and 450 mm at different locations and by different operators.

Models, Theoretical↗

PubMedDate not supplied

Simulation and experiment of temperature and cosolvent effects in reversed phase chromatography of peptides.

Experiments and simulations have been carried out for several polar protected peptides in reversed phase chromatography in order to demonstrate how simulation can describe the effects of varying temperature and cosolvent fraction. Comparisons of adsorption chemical potentials from mesoscopic simulations and experimental chromatographic retention data show very good agreement with only one temperature-independent solvent parameter from a single peptide. Such simulations should help guide the design of chromatography experiments with biomolecules and predict retention, including conditions for which empirical correlations such as hydrophobicity scales and molecular descriptors have not been developed.

Adsorption↗